The great majority of bacterial environmental responses, including many health threatening virulence strategies and antibiotic resistance mechanisms, is controlled by two-component signal transduction systems. The activating event in every two-component system is phosphOrylation of the response regulator. CheY is the prime structural model for the regulatory domain of response regulators. The CheY molecule functions in discrete steps of phosphOrylation, activation, signaling, and dephosphorylation. Models of these molecular mechanisms have been proposed based on the x-ray structures of many CheY mutants, but all models to date are limited because of lack of knowledge of the conformation of the activated, unstable form of CheY. We now have the capability of alkylating the sole cysteine of the D57C mutant CheY to produce a phosphonate moiety at the phosphorylation site Of the molecule. This modified CheY exhibits characteristics Of the phosphorylated, activated form, except that It has very long stability. This activated form of CheY has been purified and crystallized. In general, the long term objective of this proposal is to determine the mechanisms of the post- phosphorylation events of the CheY molecule through a combination of chemical modifications, and behavioral, genetic, biochemical, and structural analyses. The specific aims of this research are to answer the following questions: 1) What is the structure of CheY in the active conformation? 2) How does the inactivating effect of the T87l mutant dominate over the activation event? 3) What is the structural basis of hypersignaling in the Y1O6W mutant in the active conformation? 4) How does the T871 mutant block the otherwise hypersignaling activity of the Y1O6W mutant? 5) What is the catalytic role of the buried waters in GheY's dephosphorylation mechanism? By answering these questions, we will understand the structural basis of GheY's function. This information will be directly applicable to the mechanisms of action of response regulators in all other two-component signal transduction pathways. We are also extending the scope of this work to include molecular structure studies of other bacterial proteins which control transcriptional regulation of the flagellar gene products. Thus, our additional specific aim is: 6) Determine the structural basis of function for the master regulatory protein flhD.